Process for extracting compounds from plants

ABSTRACT

The present invention provides a method for selectively extracting acidic and/or non-acidic compounds from natural material such as plant tissue.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is a Continuation In Part application claimingbenefit under 35 U.S.C. §120 of U.S. application Ser. No. 09/969130,filed Oct. 1, 2001, which in turn claimed benefit under 35 U.S.C. 119(e)of U.S. Provisional Application Serial No. 60/236,579 filed Sep. 29,2000, both of which are incorporated herein by reference in theirentirety.

BACKGROUND OF THE INVENTION

[0002] Many plants accumulate organic substances in quantitiessufficient to be economically useful as chemical feedstocks or rawmaterials for various scientific, technological, and commercialapplications. Economically useful organic substances serve as sources ofindustrial oils, resins, tannins, saponins, natural rubber, gums, waxes,dyes, pharmaceuticals, and many specialty products.

[0003] Plant chemicals are often classified as either primary orsecondary metabolites. Primary plant metabolites are substances widelydistributed in nature, occurring in one form or another in virtually allorganisms. Secondary plant metabolites are compounds biosyntheticallyderived from primary metabolites and are more limited in distribution inthe plant kingdom. Secondary metabolites are frequently accumulated byplants in smaller quantities than are primary metabolites.

[0004] Secondary plant metabolites present a broad range of medicinalproperties. Many folk remedies are based on the isolation andpurification of secondary metabolites from trees, shrubs, and flowers.Recently, some plant secondary metabolites have been found to exhibitcancer-inhibiting activity, or other activity related to inhibitingdiseases. For example, camptothecin, colchicine, docetaxel, etopside,paclitaxel, podophyllotoxin, tetrahydrocannabinol, topotecan,vinblastine, vincristine, vindesine, betulinc acid, as well as others,have been found to have anticancer activity.

[0005] The use of secondary metabolites to treat diseases such as canceror human immunovirus (HIV) has been impeded, in part, by the difficultyassociated with synthesizing secondary plant metabolites, usingconventionally industrial chemical techniques. Because secondary plantmetabolites often have highly complex structures with many chiralcenters that may impart biological activity, such complex compoundscannot by synthesized economically. As a result, there is a need for aninexpensive, efficient, bulk method for selectively extracting secondarymetabolites from plants.

SUMMARY OF THE INVENTION

[0006] The present invention provides a method for selectivelyextracting compounds from plants in commercial (e.g., kg) quantities.The method includes contacting a mixture of a basic component and afirst solvent with plant tissue, wherein the plant tissue is optionallycontained in an extraction vessel. This contacting provides for theformation of salts with the acidic part of the plant tissue. Thiscontacting between plant tissue and basic component can be mild enoughto not cause any structural changes (by processes such as hydrolysis,oxidation, or isomerization) of the chemical sought to be extracted, orof other compounds present in plant tissue, other than removal of one ormore protons from acidic compounds or protonated basic compounds. Thecontacting should result in ionic salts between the basic pretreatmentcomponent and acidic compounds of plant tissue. A second solvent can becontacted with the plant tissue to remove non-acidic compounds. Amixture of an acidic component and a third solvent can then be contactedwith the plant tissue to remove acidic compounds, thereby providing theacidic compounds. The contact between the mixture of the third solventand acidic component with the plant tissue can also be mild enough tonot cause any structural change of the acidic compound sought to beextracted, or of the compounds present in plant tissue, other than toprotonate the basic compounds and the salts of acidic compounds.

[0007] The present invention provides for a method for selectivelyextracting one or more non-acidic compounds from plant tissue in thepresence of one or more acidic compounds, comprising: (a) contacting asolution of a basic component and a first solvent with the plant tissueto immobilize the acidic compound on the plant tissue; and (b)contacting the plant tissue with a second solvent suitable to remove thenon-acidic compounds to provide a solution comprising the non-acidiccompounds. The resulting plant tissue can optionally be contacted with asolution of an acidic component and a third solvent to remove the acidiccompounds from the plant tissue.

[0008] The present invention also provides for a method for selectivelyextracting one or more non-acidic compounds from plant tissue in thepresence of one or more acidic compounds comprising: (a) contactingplant tissue with a solution of an aluminum alkoxide in a first solventcomprising xylene, thereby effectively immobilizing the one or moreacidic compounds on the plant tissue; and (b) contacting the planttissue with a second solvent suitable to remove the one or morenon-acidic compounds, wherein the one or more non-acidic compoundscomprises lupeol, betulin, taxol, paclitaxel, echinacea extract,valerian root extract, ginkgolide A, ginkgolide B, ginkgolide C,bilobalide, garlic extract, ginseng extract, aloe gel, barbaloin,cranberry extract, eleutheroside A, eleutheroside B, eleutheroside C,eleutheroside D, eleutheroside E, eleutheroside G, kava extract, dillseed oil, kola extract, quinoline alkoloids, or a combination thereof.The method can optionally include contacting the plant tissue with asolution of acetic acid and a third solvent comprising xylene,isopropanol, or a combination thereof, to effectively remove the one ormore acidic compounds, wherein the one or more acidic compoundscomprises betulin acid, betulin-3-caffeate, tannin, lipid, phenol,caffeic acid, cichoric acid, valerenic acid, isovaleric acid, flavonoid,quercetin, kaempferol, catechin, lignin, shikimic acid, succinic acid,amino acid, nicotinic acid, pantothenic acid, anthraquinone, acidicgalactan, benzoic acid, quinic acid, malic acid, citric acid, hippuricacid, phenolic acid, ferulic acid, chlorogenic acid, norsolorinic acid,or a combination thereof.

[0009] The present invention also provides for a method for selectivelyextracting lupeol, betulin, or a combination thereof from birch bark inthe presence of a mixture of acidic compounds comprising: (a) contactingthe birch bark with a solution of an aluminum alkoxide in a firstsolvent comprising xylene, thereby effectively immobilizing the one ormore acidic compounds on the birch bark; and (b) contacting the birchbark with a second solvent suitable to remove the lupeol, betulin, or acombination thereof. The method can optionally include contacting theplant tissue with a solution of acetic acid and a third solventcomprising xylene, isopropanol, or a combination thereof, to effectivelyremove the one or more acidic compounds, wherein the one or more acidiccompounds comprises betulin acid, betulin-3-caffeate, or a combinationthereof.

[0010] The present invention also provides for the one or morenon-acidic compounds as described herein, as prepared by any one of themethods disclosed herein.

[0011] The present invention also provides for the one or more acidiccompounds as described herein, as prepared by any one of the methodsdisclosed herein.

[0012] The present invention also provides for a composition of mattercomprising the one or more acidic compounds described herein, asprepared by any one of the methods disclosed herein.

[0013] The present invention also provides for Taxol obtained by theprocess of contacting a solution of a basic component and a firstsolvent with Taxus yunnanesis bark to immobilize one or more acidiccompounds on the Taxus yunnanesis bark and contacting the Taxusyunnanesis bark with a second solvent suitable to remove the taxol,thereby providing taxol comprising less than about 5 wt. % of tannins,fatty acids, phenols, or a combination thereof.

[0014] The present invention also provides for betulin, lupeol, or acombination thereof, obtained by the process of contacting a mixture ofa basic component and a first solvent with birch bark to immobilize oneor more acidic compounds on the birch bark; and contacting the birchbark with a second solvent suitable to remove the betulin, lupeol, orthe combination thereof, thereby providing betulin, lupeol, or thecombination thereof that comprises less than about 5 wt. % of betulinicacid, betulin-3-caffeate, or a combination thereof.

BRIEF DESCRIPTION OF THE FIGURES

[0015]FIG. 1 depicts an exemplary apparatus for the use of selectivelyextracting compounds from natural materials, such as plant tissue.

DETAILED DESCRIPTION OF THE INVENTION

[0016] All plant tissue includes both acidic and non-acidic compounds.This complicates extraction processes employed to isolate non-acidiccompounds from acidic compounds of plant tissue. Therefore, the presentextraction method can be employed for the selective extraction of awide-range of plant materials. The present extraction procedure istherefore advantageous for many commercial industries, including, e.g.,pharmaceutical, cosmetic, and nutraceutical industries. For example,undesirable acidic components may be present in natural extracts alongwith desirable non-acidic compounds. These acidic components may notonly have little or no therapeutic utility, but many mammals (e.g.,humans) may have adverse reactions to these undesirable acidiccomponents. The acidic compounds, such as betulinic acid, may also bevery desirable. The method of the present invention can be used toselectively extract non-acidic components from plant tissue, wherein thenon-acidic compounds are essentially free of acidic compounds. Themethod of the present invention can also be used to selectively extractacidic compounds from plant tissue, wherein an acidic compounds areessentially free from the non-acidic compounds.

[0017] Plant Tissue

[0018] As used herein, “plant tissue” refers to the tissue of anyorganism of the plant kingdom, as opposed to one of the animal kingdomor of the kingdoms of Fungi, Protista, or Monera. The plant tissue canbe any portion or portions of the plant (e.g., bark, roots, leaves,flowers, needles, bulbs, berries, rhizomes, rootstocks, stems, andseeds), as well as the entire plant. The tissues of a plant (“planttissue”) generally fall into three main categories: dermal tissue,ground tissue, and vascular tissue. Dermal tissue refers to the “skin”layer of all plant organs and is responsible for environmentalinteraction (light passage, gas exchange, pathogen recognition andprotection, color display, etc.). Dermal tissue is composed of epidermalcells, closely packed cells that secrete a waxy cuticle that aids in theprevention of water loss. Ground tissue lies between dermal tissue andvascular tissue. The ground tissue comprises the bulk of the primaryplant body. Parenchyma, collenchyma, and sclerenchyma cells are commonin the ground tissue. In roots, the ground tissue may store sugars orstarches to fuel the spring sap flow; in leaves, the ground tissue isthe layer responsible for photosynthesis (the mesophyll). Vasculartissue transports food, water, hormones and minerals within the plant.Vascular tissue includes xylem, phloem, parenchyma, and cambium cells.

[0019] As used herein, “bark” refers to the dry, dead outer covering ofwoody branches, stems and roots of plants that is very distinct andseparable from the wood itself. It includes all tissue outside thecambium (growth layer between bark and wood).

[0020] As used here the terms “leaf” or “leaves” refer to those parts ofa plant which grow along the sides of branches or stems or at the basesof plants. Most are green and contain chlorophyll, though they vary intheir shapes and sizes. Leaves are the part of the plant that ordinarilyperforms photosynthesis (the process that converts sunlight and carbondioxide into energy).

[0021] As used herein, “needle” generally refers to a narrow stiff leaf,such as those of conifers (e.g., pine trees).

[0022] As used herein, “root” refers to the part of a plant, normallyunderground, that absorbs nutrients and anchors the plant into theground.

[0023] As used herein, “bulb” refers to a spheroidal body growing from aplant either above or below the ground (usually below), which is usuallya bud, consisting of a cluster of partially developed leaves, andproducing, as it grows, a stem above, and roots below, (e.g., the onionor tulip bulb). A true bulb is a complete package containing next year'splant (flower) already forming inside. The contents of the bulb areoften enclosed in protective, fleshy scales, which are held together bya small basal plate. The scales are modified leaves that contain enoughnutrients to sustain the plant through dormancy and early growth. Theymay be loose and open like those of a lily, or tightly closed like thoseof a hyacinth. In many bulbs, a paper-thin tunic protects the scales(lilies don't have a tunic). Roots will grow from the bulb's basalplate.

[0024] As used herein, “berry” refers to any small fruit that is pulpyor succulent throughout, having seeds loosely imbedded in the pulp, suchas the currant, grape, or blueberry. Berry can be further defined as anindehiscent fruit derived from a single ovary and having the whole wallfleshy, such as the grape or tomato. Furthermore, berries come invarious structures including simple, such grape; blueberry, cranberry,or aggregate, such as blackberry; raspberry, strawberry mulberry.

[0025] As used herein, “rhizome” refers to a horizontal, usuallyunderground stem that often sends out roots and shoots from its nodes(also called rootstalk or rootstock).

[0026] As used herein, “rootstock” refers to a robust plant thatprovides the root system in grafting, also known as a stock. Scions andbuds are grafted and budded to a rootstock or stock. Rootstock alsorefers to the elongated and often thick rhizomes of certain perennialherbaceous plants such as the Iris, Aspidistra and Solomon's Seal.

[0027] As used herein, “stem” refers to the main (usually aerial) axis(sometimes referred to as the trunk or stalk) of a tree, shrub, orplant. “Stem” also refers to the part of the plant that supports theleaves, flowers or fruits of a plant, such as the peduncle of a fruit orthe pedicel of a flower.

[0028] As used herein, “seed” refers to a ripened ovule, consisting ofan embryo with one or more integuments, or coverings, such as an appleseed, a currant seed, dill seed, or kola nut seed. By germination, mostseeds produces a new plant. “Seed” also refers to any small seedlikefruit, though it may consist of a pericarp, or even a calyx, as well asthe seed proper, such as a parsnip seed or thistle seed. The seed properhas an outer and an inner coat, and within these the kernel or nucleus.The kernel is either the embryo alone, or the embryo enclosed in thealbumen, which is the material for the nourishment of the developingembryo. The scar on a seed, left where the stem parted from it, iscalled the hilum, and the closed orifice of the ovule, the micropyle.

[0029] Plant

[0030] The plant can be a bryophyte or vascular plant. Morespecifically, the plant can be grass, flower or a tree and the planttissue can be any part of the grass, flower or tree. Specific plants,flowers, and trees include, e.g., Moss (e.g., Club Moss), Horsetail,Fern, Conifer, Cycad, Ginkgo biloba (Ginkgo), Taxus yunnanesis (yewtree), Echinacea spp., Valeriana officinalis, Allium sativum (garlic),Panax ginseng, aloe vera, Vaccinium macrocarpon, Eleutherococcussenticosus, Piper methysticum, dill, kola nut, and cinchona.

[0031] Another specific plant is the birch tree, wherein the suitableplant tissue can be the bark of the birch tree. As used herein, “birch”or “birch tree” refers to any of the several deciduous tress of thegenus Betula. The birches comprise the family Betulaceae in the orderFagales. Birch trees include, for example, white birch, B. alba; sweet,black or cherry birch, B. lenta; monarch birch, B. maximowicziana; dwarfor arctic birch; B. nana; Japanese white birch, B. platphylajaponica;smooth-bark birch, B. pubescens; yellow birch, B. alleghaniensis; paper,white or canoe birch, B. papyrifera; gray birch, B. populifolia; riverbirch, B. nigra; and the European birches, B. pubescens; B. alba and B.pendula. Specifically, birch can be B. alba, B. lenta, B.maximowicziana, B. nana, B. platyphyla japonica, B. pubescens, B.alleghaniensis, B. papyrifera, B. populifolia, B. nigra or B. pendula. Aspecific birch for use in the processes of the present invention is B.papyrifera.

[0032] As used herein, “Taxus” or “yew” refers to plants belonging toTaxaceae Gymnopemnae. There are 11 species and five sub-species of Taxusin the world, mainly found in East Asia, North America, and Europe;

[0033] “Echinacea spp.” refers to a perennial native to North Americanwhich resembles a black-eyed Susan and is called echinacea, purpleconeflower or snake root;

[0034] “Valeriana officinalis” or “valerian” refers to the plantValeriana officinalis of the valerianaceae family, which is also knownas valerian, phu, all-heal, great wild valerian, amantilla, setwall,setewale, capon's tail;

[0035] “Allium sativum” refers to garlic;

[0036] “Panax ginseng” refers to ginseng, commonly called Koreanginseng, Chinese ginseng or American ginseng. Asian ginseng is a memberof the Araliaceae family, which also includes the closely relatedAmerican ginseng, Panax quinquefolius, and less similar Siberianginseng;

[0037] “Eleutherococcus senticosus,” refers to “eleuthero” (whichcontains eleutheroside A, eleutheroside B (syringin), eleutheroside C,eleutheroside D, eleutheroside E (syringaresinol di-O-β-D-glucoside,liriodendrin), and eleutheroside G, among other constituents);

[0038] “Aloe” refers to any of the over 500 different species of Aloe.Aloe Vera is a member of the Lily family and is very-cactus like in itscharacteristics. This unique plant also belongs to a larger plant familycalled “Xeroids”. Of the 500+ species of Aloe, Aloe barbadensis miller(Aloe Vera species) is preferred;

[0039] “Vaccinium macrocarpon” refers to cranberry;

[0040] “Piper methysticum,” a member of the pepper family, refers to aplant native to the South Sea Islands of Micronesia, Melanesia andPolynesia;

[0041] “Kola vera,” of the family N.O. Sterculiaceae, also known as“Kola nut” refers to the tree that grows about 40 feet high and hasyellow flowers spotted with purple; and

[0042] “Cinchona,” belongs to the family N.O. Rubiaceae and refers toPeruvian bark (Cinchona succirubra) which is an evergreen tree thatgrows 15 to 45 feet in height.

[0043] Plant Components (Non-Acidic Compounds and Acidic Compounds)

[0044] The specific non-acidic compounds and acidic compounds that canbe isolated from the plant tissue will depend, in part, upon thespecific plant tissue that is being extracted. For example, the bark ofTaxus yunnanesis can be extracted employing the methods described hereinto provide taxol (paclitaxel) as the non-acidic compound and tannin,fatty acids, and phenols as the acidic compounds;

[0045] the needles of the Yew tree can be extracted employing themethods described herein to provide taxol (paclitaxel) as the non-acidiccompound, and tannin, fatty acids, and phenols as the acidic compounds;

[0046] the root of the Echinacea spp. can be extracted employing themethods described herein to provide Echinacea extract as the non-acidiccompound and tannin, caffeic acid, and cichoric acid as the acidiccompounds;

[0047] the root of the Valeriana officinalis can be extracted employingthe methods described herein to provide Valerian root extract as thenon-acidic compound and valerenic acid, isovaleric acid, and tannins asthe acidic compounds;

[0048] the roots, bark, leaves, or any combination thereof of the Ginkgobiloba can be extracted employing the methods described herein toprovide Ginkgolide A, Ginkgolide B, Ginkgolide C, and bilobalide as thenon-acidic compounds and tannins, flavonoids (e.g., quercetin,kaempferol, catechin), lignins, shikimic, and succinic acids as theacidic compounds;

[0049] the bulb of the Allium sativum can be extracted employing themethods described herein to provide garlic extract as the non-acidiccompound and fatty acids and amino acids as the acidic compounds;

[0050] the root of the Panax ginseng can be extracted employing themethods described herein to provide Ginseng extract as the acidiccompound and tannin, fatty acids, nicotinic acid and pantothenic acid asthe acidic compound;

[0051] the leaves of the Aloe Vera can be extracted employing themethods described herein to provide aloe gel and barbaloin as thenon-acidic compounds and fatty acids, anthraquinones, acidic gelactan,and amino acids as the acidic compounds;

[0052] the berries of the Vaccinium macrocarpon can be extractedemploying the methods described herein to provide cranberry extract asthe non-acidic compounds and benzoic acid, quinic acid, malic acid,citric acid, and hippuric acid as the acidic compounds;

[0053] the roots, rhizomes, stems, leaves, or combination thereof of theEleutherococcus senticosus can be extracted employing the methodsdescribed herein to provide Eleutherosides A-G as the non acidiccompounds and tannin, fatty acids, and caffeic acid as the acidiccompounds;

[0054] the rootstock of the Piper methysticum can be extracted employingthe methods described herein to provide Kava extract as the non-acidiccompounds and tannin, fatty acids, and amino acids as the acidiccompounds;

[0055] the seeds of the Dill can be extracted employing the methodsdescribed herein to provide seed oil as the non-acidic compound andphenolic acids (caffeic acid, ferulic acid, and chlorogenic acid) as theacidic compounds;

[0056] the seeds of the Kola nut can be extracted employing the methodsdescribed herein to provide kola extract as the non-acidic compounds andtannin and catechins as the acidic compounds; and

[0057] the bark of the cinchona (yellow or red) can be extractedemploying the methods described herein to provide quinolone alkaloids asthe non-acidic compounds and norsoloric acid, tannins, and quinic acidas the acidic compounds. TABLE 1 Non-acidic compounds and acidiccompounds that can be isolated from specific plant tissue. Components ofInterest Acidic Plant Tissue (non-acidic) Components Taxus yunnanesisBark Taxol (paclitaxel) Tannin, fatty acids, phenols Yew tree NeedlesTaxol (paclitaxel) Tannin, fatty acids, phenols Echinacea spp. RootEchinacea extract Tannin, caffeic, cichoric acid, tartaric acid &dicaffeate Valeriana Root Valerian Roots Valerenic acid, officinalisextract Isovaleric acid, tannins Ginkgo biloba Root bark Ginkgolide A, BTannins, and leaves and C, bilobalide flavonoids (quercetin, kaempferol,catechin), lignins, shikimic and succinic acids Allium sativum BulbGarlic extract Fatty acids, (garlic) amino acids Panax ginseng RootGinseng extract Tannin, fatty acids, nicotinic acid, pantothenic acidAloe Vera Leaves Aloe gel, Fatty acids, barbaloin anthraquinones, acidicgalactan, amino acids Vaccinium Berry Cranberry extract benzoic,macrocarpon quinic, malic, citric and hippuric acid EleutherococcusRoot, rhizome, Eleutherosides Tannin, fatty senticosus stems, leaves A-Gacids, caffeic acid Piper methysticum Rootstock Kava extract Tannin,fatty acids, amino acids Dill Seeds Seed oil Phenolic acids (caffeic,ferulic, chlorogenic) Kola nut Seeds Kola extract Tannin, catechinsCinchona (red and Bark Quinoline Cinnamic acid, yellow) alkaloidstannins, quinic acid

[0058] “Paclitaxel” refers to[2aR-[2aα,4β,4aβ,6β,9α(αR*,βS*),-11,α12α,12aα,12bα]]-β-(Benzoylamino)-α-hydroxybenzenepropanoicacid 6,12b-bis(acetyloxy)-12-(benzoyloxy)-2a,3,4,4a,5,6,9,10,11,12,12a,12b-dodecahydro-4,11,-dihydroxy-4a,8,13,13-tetramethyl-5-5oxo-7,11-methano-1H-cyclodeca[3,4]benz[1,2-b]oxet-9-yl ester.

[0059] “Echinacea extract” is believed to include essential oil,polysaccharides, such as inulin, polyacetylenes, betain, glycoside,sesquiterpenes and caryophylene. Echinacea extract is also believed tocontain copper, iron, tannins, protein, fatty acids, fat-solublealkylamides, caffeic acid glycoside (echinacoside), and vitamins A, C,and E.

[0060] “Valeriana officinalis extract” is a very effective sedative andis used most often to help insomnia, especially due to stress. It has anadvantage over prescription sedatives in that it is not habit forming.Valerian has many actions besides its well-known sedative effects. Itstrengthens the heart and in some cases lower blood pressure. Itpromotes wound healing and has some antibiotic activity and may be usedexternally to relieve muscle cramps. It has some expectorant activityand may help a tickly cough. It may actually balance the nervous systemhelping to calm agitated states and stimulate cases of extreme fatigue.There are several species of valerian, which vary in potency and can beused similarly, although V. officinalis is the preferred plant. Otherconstituents are a volatile oil, which includes isovalerianic acid andborneol; choline; flavonoids; sterols and several alkaloids, includingactinidine, valerianine, valerine, and chatinine. Valepotriates are notwater-soluble, but valeric acid is and may be the constituent mostlikely to produce valerian's sedative effect, especially when used as itwas traditionally in water extracts (teas) or water/alcohol extracts(tinctures).

[0061] Many studies have provided clinical evidence that ginkgo preventsmany problems throughout the entire body. Ginkgo is gaining recognitionas a brain tonic that enhances memory because of its positive effects onthe vascular system, especially in the cerebellum. It is also used as atreatment for vertigo, tinnitus (ringing in the ears) and a variety ofneurological disorders and circulation problems. Ginkgo may help tocounteract the effects of aging, including mental fatigue and lack ofenergy. Ginkgo has two groups of active substances, flavonoids (athree-ringed molecule with hydroxyl (OH) groups attached) and terpenelactones, including ginkgolides A, B, and C, bilobalide (asesquiterpene), quercetin (a flavonoid), and kaempferol (a flavonoid).The constituents of gingko include terpenoids (bilobalide), diterpenoids(ginkgolide A, ginkgolide B, ginkgolide C, ginkgolide J, ginkgolide M),flavonoids: flavones (luteolin, tricetin, 2-hydroxyluteolin), biflavones(amentoflavone, ginkgetin, isoginkgetin, sciadoptysin,5-methoxybilobetin, bilobetin), flavonols (caempherol, quercetin,isorhammetin), flavone glycosides, flavone acyl glycosides, catechins,and steroids (sitosterol, sitosterol glucoside). The ginkgolides havebeen shown to control allergic inflammation, anaphylactic shock andasthma. Ginkgo extract is generally derived from dried ginkgo leaves,but also may be derived from gingko root or bark.

[0062] “Garlic” contains compounds that are antibacterial, antifungaland reduce blood clotting. In order for the active ingredient that givesgarlic its characteristic odor and its therapeutic effects to bereleased, the garlic clove (or bulb) must be cut or crushed. Thisreleases an enzyme that causes the formation of allicin, the componentresponsible for garlic's odor and medicinal activity. Activeconstituents present in garlic include the sulphur compound allicin,produced by crushing or chewing fresh garlic, which in turn producesother sulphur compounds: ajoene, allyl sulfides, and vinyldithiins.

[0063] “Ginseng” is believed to increase energy, counter the effects ofstress, and enhance intellectual and physical performance. Thirteenginsenosides have been identified in ginseng, including ginsenosides Rg1and Rb1. Other constituents include the panaxans, which are believed tohelp lower blood sugar, and the polysaccharides (complex sugarmolecules), which are believed to support immune function. Also,long-term intake may be linked to a reduced risk of cancer.

[0064] Applied to wounds, “aloe” is a mild anesthetic, relievingitching, swelling, and pain: it also is antibacterial and antifungal,increases blood flow to wounded areas, and stimulates fibroblasts, theskin cells responsible for wound healing.

[0065] “Cranberry” has astringent applications for the urinary tract andis a traditional remedy for bladder infections and kidney-relateddisorders. Two components of cranberry juice have been shown to inhibitthe adherence of E. coli to uroepithelial cells. The first is fructose.The second is proanthocyanidin, the chemical structure of which has beenelucidated. Fructose inhibits the adherence of type-1 fimbriated E. coliand proanthocyanidin inhibits the adherence of P-fimbriated E. coli touroepithelial cells. Cranberry is also a natural diuretic and urinaryantiseptic agent.

[0066] Although “kava” has undergone much research as to its activeingredients, there is still no definite conclusion. It consists of anoleoresin from which kavalactones originate, starch, sugars, proteins,vitamins B1, B2, B3, B6, folic acid and E, potassium, manganese, biotin,choline, inositol, fat, glycyrrhizin, lecithin, pantothenic acid,para-aminobenzoic acid, pentacyclic terpenes, phosphorous, and a yellowdye. Kavalactones are considered the most active constituents in theplant. The main use for kava today is in the treatment of anxiety. It isalso an excellent muscle relaxant and has diuretic and urinaryantiseptic properties, so it may be useful in urinary cystitis andprostatitis. Kava also shows pain-relieving properties.

[0067] “Kola vera” or “Cola vera” seeds are said to contain a glucoside,Kolanin (this substance may be a mixture of Kola red and caffeine). Theseeds also contain starch, fatty matter, sugar, and a fat decomposingenzyme acting on various oils.

[0068] “Dill seed” is an herbal medicine that is used to reduce gas,upset stomach, and colic pains. It is also used to promote the flow ofmilk in breastfeeding mothers, and to help control bad breath andhiccups. Other names for Dill Seed include: Anethum Graveolens, Dill,and Dillweed.

[0069] As used herein, “tannin” refers to tannic acid or gallotannicacid. Tannin varies somewhat in composition, depending on the source,having the approximate empirical formula C₇₆H₅₂O₄₆. Tannic acid is acolorless to pale yellow solid; it is believed to be a glucoside inwhich each of the five hydroxyl groups of the glucose molecule isesterified with a molecule of digallic acid. Tannin is used in tanninganimal skins to make leather; it transforms certain proteins of animaltissue into compounds that resist decomposition. It is also used inmanufacturing inks, as a mordant in dyeing, and in medicine as anastringent and for treatment of bums.

[0070] As used herein, “fatty acids” refers to a long-chain ofcarboxylic acids that may either be saturated (without double bond) ornon-saturated (with double bond). It refers to any acid derived fromfats by hydrolysis (e.g., oleic acid, palmitic acid, or stearic acid);any long-chain monobasic organic acid.

[0071] As used herein, “phenols” refers to compounds that include aC₆H₅OH backbone. They are aromatic alcohols that are optionallysubstituted by one or more substituents. Phenols exhibits weak acidicproperties and are sometimes called carbolic acids, especially when inwater solution.

[0072] As used herein, “caffeic acid” refers to3-(3,4-Dihydroxyphenyl)-2-propenoic acid.

[0073] As used herein, “valeric acid” refers to pentanoic acid;valerianic acid; and propylacetic acid.

[0074] As used herein, “isovaleric acid” refers to 3-Methylbutanoic acidand isovalerianic.

[0075] As used herein, “flavonoid” refers to polyphenols that have acarbon skeleton. They have an acidic nature due to the phenol groups.

[0076] As used herein, quercetin refers to2-(3,4-Dihydroxyphenol)-3,5,7-trihydroxy-4H-1-benzopyran-4-one.

[0077] As used herein, “kaempferol” refers to3,5,7-Trihydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one.

[0078] As used herein, “catechin” refers to(2R-trans)-2-(3,4-dihydroxyphenyl)-3,-4-dihydro-2H-1-benzopyran-3,5,7-triol.

[0079] As used herein, “lignin” refers to a highly polymerized andcomplex chemical compound especially common in woody plants. Thecellulose walls of the wood become impregnated with lignin, a processcalled lignification, which greatly increases the strength and hardnessof the cell and gives the necessary rigidity to the tree. It isessential to woody plants for them to stand erect.

[0080] As used herein, “amino acids” refers to any one of a class ofsimple organic compounds containing carbon, hydrogen, oxygen, nitrogen,and in certain cases sulfur. These compounds are the building blocks ofproteins. They are characterized by the presence of a carboxyl group(COOH) and an amino group (NH₂). The 20 amino acids commonly found inanimals are alanine, arginine, asparagine, aspartic acid, cysteine,glutamic acid, glutamine, glycine, histidine, isoleucine, leucine,lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, and valine. More than 100 less common amino acidsalso occur in biological systems, particularly in plants. Every aminoacid except glycine can occur as either of two optically activestereoisomers, D or L; the more common isomer in nature is the L-form.When the carboxyl carbon atom of one amino acid covalently binds to theamino nitrogen atom of another amino acid with the release of a watermolecule, a peptide bond is formed.

[0081] As used herein, “shikimic acid” refers to[3R-(3α,4α,5β]-3,4,5-Trihydrooxy-1-cyclohexene-1-carboxylic acid.

[0082] As used herein, “succinic acid” refers to butanedoic acid(HOOCCH₂CH₂COOH).

[0083] As used herein, “nicotinic acid” refers to 3-Pyridinecarboxylicacid.

[0084] As used herein, “pantothenic acid” refers to“(R)-N-(2,4-Dihydroxy-3,3-dimethyl-1-oxobutyl)-β-alanine.

[0085] As used herein, “anthraquinone” refers to 9,10-anthracenedione.

[0086] As used herein, “acidic galactan” refers to a poly sugar withattached carboxylic groups.

[0087] As used herein, “benzoic acid” refers to benzoic acid, C₆H₅CO₂H.It is the simplest aromatic carboxylic acid. In addition to beingsynthesized from a variety of organic compounds (e.g., benzyl alcohol,benzaldehyde, toluene, and phthalic acid), it may be obtained fromresins, notably gum benzoin. It is used largely for making its salts andesters, most notably sodium benzoate, which is widely used as apreservative in foods and beverages and as a mild antiseptic inmouthwashes and toothpastes.

[0088] As used herein, “quinic acid” refers to[1R-(1α,3α,4α,5β]-1,3,4,5-Tetrahydroxycyclohexanecarboxylic acid.

[0089] As used herein, “malic acid” refers to hydroxybutanedioic acid.

[0090] As used herein, “citric acid” refers to citric acid or2-hydroxy-1,2,3-propanetricarboxylic acid, HO₂CCH₂C(OH)(CO₂H)CH₂CO₂H, anorganic carboxylic acid containing three carboxyl groups. It isresponsible for the tart taste of various fruits in which it occurs,e.g., lemons, limes, oranges, pineapples, and gooseberries.

[0091] As used herein, “hippuric acid” refers to N-Benzoylglycine.

[0092] As used herein, “ferulic acid” refers to3-(4-Hydroxy-3-methoxyphenyl)-2-propenoic acid.

[0093] As used herein, “chlorogenic acid” refers to[1S-(1α,3β,4α,5α]-3-[[3-(3,4-Dihydroxyphenyl)-1-oxo-2-propenyl]oxy]-1,4,5,trihydroxycyclohexanecarboxylicacid.

[0094] As used herein, “cinnamic acid” refers to 3-phenyl-2-propenoicacid.

[0095] Extraction Procedures

[0096] As used herein, “extraction” refers to a technique for separatinga mixture of chemical components from plant tissue, wherein thecomponents that are separated have different solubilities and adsorptionstrengths. A “solvent extraction” is a type of extraction wherein amixture of components adsorbed on plant tissue are separated utilizingthe differences in the solubilities and adsorption strengths of thecomponents that are separated. Suitable extraction techniques aredisclosed, e.g., in “Experiments in Organic Chemistry: From Microscaleto Macroscale,” Jonathan S. Nimitz (New York: Prentice Hall, 1990).

[0097] As used herein. “selective extraction” refers to the process ofextracting a class of one or more compounds (e.g., one or morenon-acidic compounds) from another class of one or more compounds (e.g.,one or more acidic compounds).

[0098] As used herein, “acidic compound” in plant tissue refers to anycompound naturally found in plant tissue that is acidic enough to formsalts with the basic components upon treatment with the basiccomponents. For instance, plant phenols, flavonoids, flavones,flavolonoles, and tannins are acidic enough to form salts that areimmobilized on plant tissue by treatment with the basic components.

[0099] As used herein, an “acid” refers to any compound or mixture ofcompounds, in any suitable and effective amount, that can effectivelylower the pH of a neutral solution to below 7.0. The acid will act as aproton donor and can neutralize a basic component or solution of basiccomponents, thereby forming a salt and water. Any suitable acid can beemployed, provided the acid effectively neutralizes the one or moresalts, which are formed after the plant tissue immobilization. Thesuitable acid may be an inorganic acid (e.g., hydrochloric acid,hydrobromic acid, sulfuric acid, phosphoric acid, or a combinationthereof); an organic acid (e.g., acetic acid, formic acid, or acombination thereof); or a combination of an inorganic acid and anorganic acid.

[0100] As used herein, a “basic component” refers to any compound ormixture of compounds, in any suitable and effective amount, that caneffectively form non-soluble salts with the one or more acidic compoundsand effectively immobilizes the one or more acidic compounds on theplant tissue. The basic component will act as a proton acceptor. Anysuitable basic component can be employed, provided the basic componenteffectively forms non-soluble salts with the one or more acidiccompounds and effectively immobilizes the one or more acidic compoundson the plant tissue. One suitable class of basic components thateffectively forms non-soluble salts with the one or more acidiccompounds and effectively immobilizes the one or more acidic compoundson the plant tissue are the alcoholates.

[0101] As used herein, an “alcoholate” or “alkoxide” refers to a baseformed from an alcohol in which the hydroxyl hydrogen atom has beenreplaced by a metal atom (e.g., sodium, lithium, potassium, calcium, oraluminum). One suitable alcoholate includes the aluminum alcoholates.

[0102] As used herein, an “aluminum alcoholate” refers to an alcoholateor alkoxide in which the metal atom is aluminum. Suitable aluminumalcoholates include compounds of the formula Al(OR)₃, wherein each R isindependently (C₁-C₁₂)alkyl, aryl (e.g., phenyl), or arylalkyl (e.g.,benzyl), wherein each alkyl, aryl, or arylalkyl can be optionallysubstituted on carbon with one or more hydroxy, halo, or —N(R_(b))₂.Each R_(b) can idependently be H, (C₁-C₆)alkyl, aryl (e.g., phenyl), orarylalkyl (e.g., benzyl). Suitable specific aluminum alcoholatesinclude, e.g., aluminum isopropoxide [Al(i-OPr)₃], aluminum ethoxide[Al(OEt)₃], and aluminum methoxide [Al(OMe)_(3].)

[0103] As used herein, “alkyl” can be straight-chain or branched.

[0104] Other suitable alcoholates include sodium alcoholates (NaOR),lithium alcoholates (LiOR), potassium alcoholates (KOR), magnesiumalcoholates [Mg(OR)₂], calcium alcoholates [Ca(OR)₂], and germaniumalcoholates [Ge(OR)₃]; wherein each R is independently (C₁-C₁₂)alkyl,aryl (e.g., phenyl), or arylalkyl (e.g., benzyl), wherein each alkyl,aryl, or arylalkyl can be optionally substituted on carbon with one ormore hydroxy, halo, or —N(R_(b))₂. R_(b) is H, (C₁-C₆)alkyl, aryl (e.g.,phenyl), or arylalkyl (e.g., benzyl). Specific examples of alcoholatesinclude sodium methoxide, sodium ethoxide, potassium ethoxide, potassiumtert-butoxide, and dimethoxymagnesium.

[0105] Another suitable class of basic components includes amines. Asused herein, “amines” includes ammonia, as well as primary (NH₂R),secondary (NHR₂), and tertiary (NR₃) amines. Each R can independently be(C₁-C₁₂)alkyl, aryl (e.g., phenyl), or arylalkyl (e.g., benzyl); whereineach alkyl, aryl, or arylalkyl can be optionally substituted on carbonwith one or more hydroxy, halo, or —N(R_(b))₂. Each R_(b) canidependently be H, (C₁-C₆)alkyl, aryl, or arylalkyl. Specific examplesof amines are ammonia, triethylamine, trimethylamine, N(CH₂CH₂OH)₃, and(HOCH₂)₃CNH₂.

[0106] Another suitable class of basic components includes heterocycles.As used herein, “heterocycle” refers to an aromatic or non-aromaticcompound that contains in the ring at least one basic nitrogen atom. Aheterocyclic ring system can be simple, ortho-fused, or bicyclic. Thering system can optionally comprise one or more non-peroxide oxygen orsulfur. Examples of heterocycles include pyridine, morpholine,piperidine, N-methylpiperidine, pyrrole, pyrrolidine,azabicyclo[2.2.2]octane, and diazabicyclo[2.2.2]octane. The heterocylering system can optionally be subtituted on carbon with one or more oxo,hydroxy, amino, sulfo, (C₁-C₄)alkyl, (C₁-C₄)hydroxyalkyl, or —N(R_(b))₂,wherein R_(b) is H or (C₁-C₄)alkyl; or on nitrogen with one or more(C₁-C₄)alkyl or (C₁-C₄)hydroxyalkyl.

[0107] As used herein, “hydroxyalkyl” can be straight-chain or branched,and the hydroxy group can be on any suitable carbon atom.

[0108] Another suitable class of basic components includes alkalineearth metal hydroxides. These comprise an alkaline earth cation and oneor more hydroxide ions. Examples of alkaline earth metal hydroxidesinclude NaOH, KOH, LiOH, Mg(OH)₂, and Ca(OH)₂.

[0109] Another suitable class of basic components includes alkalineearth metal oxides. These compounds consist of one or more alkalineearth metals and oxygen. Examples of basic oxides include K₂O, Na₂O,Li₂O, KNaO, CaO, and MgO.

[0110] Another suitable class of basic components includes alkalineearth metal carbonates and bicarbonates. The compounds consist of CO₃ ²⁻or HCO₃ ⁻ and alkaline earth metal cations. Examples of alkaline earthmetal carbonates and bicarbonates include Na₂CO₃, NaHCO₃, K₂CO₃, KHCO₃,KNaCO₃, Li₂CO₃, LiHCO₃, CaCO₃, and MgCO₃.

[0111] Another suitable class of basic components includes alkalineearth metal sulfites. These compounds consist of sulfite anion andalkaline earth metal cations. Examples of alkaline earth metal sulfitesinclude Na₂SO₃, K₂SO₃, KNaSO₃, Li₂SO₃, CaSO₃, and MgSO₃.

[0112] Another suitable class of basic components includes alkalineearth metal sulfides. These compound consist of S²⁻ and alkaline earthmetal cations. Examples of alkaline earth metal sulfides include Li₂S,K₂S, and Na₂S.

[0113] Another suitable class of basic components includes alkalineearth metal hydrogen sulfides. These compounds consist of HS⁻ and analkaline earth metal cation. Examples of alkaline earth metal hydrogensulfides include LiHS, KHS, and NaHS.

[0114] The basic component can also be generated in a mixture fromcompounds that generate a basic component. Examples of compounds thatcan generate basic components would be elemental sodium, elementalmagnesium, elemental potassium, and elemental calcium. Each of thesecompounds in contact with water or alcohols will generate thecorresponding alkline earth metal hydroxide or alkaline earth metalalcoholate.

[0115] Suitable basic components are commercially available from, e.g.,Aldrich (Milwaukee, Wis.)

[0116] The first solvent can effectively dissolve the basic componentbut will not effectively dissolve the salts that are formed in theprocess of the neutralization of acidic compounds. Any suitable solventcan be employed as the first solvent, provided the solvent effectivelydissolves the basic component but does not effectively dissolve thesalts that are formed in the process of the neutralization of acidiccompounds. The first solvent can include any suitable: (1) optionallysubstituted aromatic compound, (2) optionally substituted heterocycliccompound, (3) optionally substituted cyclic compound, (4) optionallysubstituted linear or branched compound, (5) or any combination thereofSuitable substituents include, e.g., (C₁-C₆)alkyl, hydroxyl, halo,trihalo(C₁-C₆)alkyl, cyano, nitro, oxo, thioxo, amino, carboxyl, andcombinations thereof. Compounds suitable as a first solvent aredisclosed and commercially available from, e.g., 2001 Aldrich Catalogue(Milwaukee, Wis.). Specific compounds suitable as a first solventinclude isopropanol, ethanol, methanol, methylene chloride, toluene,xylene (e.g., o-xylene, m-xylene, or p-xylene), carbon dioxide, orcombinations thereof. Other compounds suitable as a first solventinclude Xe, Freon-23, ethane, N₂O, SF₆, propane, ammonia, n-C₄H₁₀,(C₂H₅)₂O, and combinations thereof. The first solvent can include asingle compound or can include a mixture of compounds. In addition, thefirst solvent can optionally include an additive.

[0117] The concentration of the basic component in the first solvent canvary depending on the carrying capacity of the solvent for the basiccomponent. Any suitable solvent can be employed that allows forefficient reaction between the basic component and acidic compounds inthe plant tissue. Typically, the concentration can be about 0.1 to 25percent basic component in the solvent by weight. Specifically, theconcentration can be about 0.5 percent to 10 percent base in the solventby weight. More specifically, the concentration can be about 1 percentto 5 percent base in the solvent by weight.

[0118] By treating the plant tissue with a mixture of a basic componentin a first solvent, acidic compounds in the plant tissue will formsalts. The resulting salts can precipitate on the plant tissue, orotherwise adhere to the plant tissue, so that non-acidic compounds canbe selectively removed from the plant tissue. Discharging the mixture ofthe basic component in the first solvent from the extraction vessel andintroducing a second solvent can accomplish this. The excess basiccomponent can be adsorbed by any suitable adsorbent (e.g., silica,alumina, or a combination thereof).

[0119] The second solvent can effectively dissolve the one or morenon-acidic compounds but will not effectively dissolve the salts of theone or more acidic compounds that were effectively formed during thetreatment with the basic component. The second solvent can include anysuitable: (1) optionally substituted aromatic compound, (2) optionallysubstituted heterocyclic compound, (3) optionally substituted cycliccompound, (4) optionally substituted linear or branched compound, (5) orany combination thereof. Suitable substituents include, e.g.,(C₁-C₆)alkyl, hydroxyl, halo, trihalo(C₁-C₆)alkyl, cyano, nitro, oxo,thioxo, amino, carboxyl, and combinations thereof. Compounds suitable asa second solvent are disclosed and commercially available from, e.g.,2001 Aldrich Catalogue (Milwaukee, Wis.). Specific compounds suitable asa second solvent include isopropanol, ethanol, methanol, methylenechloride, toluene, xylene (e.g., o-xylene, m-xylene, or p-xylene),carbon dioxide, or combinations thereof. Other compounds suitable as asecond solvent include Xe, Freon-23, ethane, N₂O, SF₆, propane, ammonia,n-C₄H₁₀, (C₂H₅)₂O, and combinations thereof.

[0120] The second solvent can include a single compound or can include amixture of compounds. In addition, the second solvent can optionallyinclude an additive. The second solvent can be passed through the vesselto remove non-acidic compounds from the plant tissue in one pass or inmultiple passes. Optionally, the second solvent can be recirculatedthrough the vessel using the reservoir optionally attached to thevessel. Non-acidic compounds can be extracted from the plant tissueusing temperature, pressure, and time parameters that are sufficient toremove a significant amount (e.g., more than about fifty percent, morethan about seventy percent, or more than about ninety percent) ofnon-acidic compounds can be removed from the plant tissue. An optionaladditional vessel can be employed for the adsorption of any excess basicmaterial and some polymeric non-acidic compounds.

[0121] The third solvent can effectively neutralize the basic salts thatare formed during the treatment of plant tissue with the basiccomponents. This process releases acidic compounds for furtherextraction with the third solvent. The third solvent can include anysuitable: (1) optionally substituted aromatic compound, (2) optionallysubstituted heterocyclic compound, (3) optionally substituted cycliccompound, (4) optionally substituted linear or branched compound, (5) orany combination thereof. Suitable substituents include, e.g.,(C₁-C₆)alkyl, hydroxyl, halo, trihalo(C₁-C₆)alkyl, cyano, nitro, oxo,thioxo, amino, carboxyl, and combinations thereof. Compounds suitable asa third solvent are disclosed and commercially available from, e.g.,2001 Aldrich Catalogue (Milwaukee, Wis.). Specific compounds suitable asa third solvent include isopropanol, ethanol, methanol, methylenechloride, toluene, xylene (e.g., o-xylene, m-xylene, or p-xylene),carbon dioxide, or combinations thereof. Other compounds suitable as athird solvent include Xe, Freon-23, ethane, N₂O, SF₆, propane, ammonia,n-C₄H₁₀, (C₂H₅)₂O, and combinations thereof. The third solvent caninclude a single compound or can include a mixture of compounds. Inaddition, the third solvent can optionally include an additive.

[0122] The solution of the acid in the third solvent can be passedthrough the vessel in one pass or in multiple passes using temperature,pressure, and time parameters that are sufficient to remove asignificant amount (e.g., more than about fifty percent, more than aboutseventy percent, or more than ninety percent) of acidic compounds fromthe plant tissue.

[0123] As used herein, an “additive” is a compound added to the solventin an amount of about 1 wt % to about 20 wt. % based on the solvent.Specifically, the additive may be present in an amount of about 1 wt. %to about 15 wt. % or about 1 wt. % to about 10 wt. %. Upon addition, theadditive will modify the physical properties of the solvent. Forexample, an additive may be useful to modify the polarity, criticaltemperature, critical pressure, etc., of the solvent system. Suitableadditives include lower alcohols (e.g., methanol, ethanol, 1-propanol,2-propanol, 1-hexanol, or 2-methoxy ethanol); ethers (e.g.,tetrahydrofuran or 1,4-dioxane); substituted hydrocarbons (e.g.,acetonitrile, dichloromethane, ammonia or chloroform) propylenecarbonate, N,N-dimethylacetamide; dimethyl sulfoxide; carboxylic acids(e.g., formic acid); water; carbon disulfide; lower ketones (e.g.,acetone), hydrocarbons (e.g., propane, toluene, hexanes and pentanes);as well as optionally substituted aromatic compounds (e.g., o-xylene,m-xylene, p-xylene, and toluene).

[0124] As used herein, “fragmentation” includes chopping, crunching,crushing, gnashing or pounding. Such fragmentation of plant tissue willeffectively provide smaller pieces of plant tissue. The smaller piecesof plant tissue will have, combined, a larger surface area. Thefragmentation can conveniently be carried out, e.g., by introducingplant tissue into a machine with knives on a rotating disk (e.g., achipper or shredder). One chipper suitable for fragmenting the planttissue is the YardMan Model 246-648D401 chipper.

[0125] As used herein, “pelletization” refers to the process of formingplant tissue pellets. Any suitable pelletization method known to thoseof skill in the art can be employed. For example, fragmented planttissue can be sprayed with a solvent (e.g., water) through a sprinklerin a horizontal mixer. Pelletization can routinely be performed using,e.g., a Laboratory Pellet Machine (California Pellet Mill, Co., Calif.)through a die with holes. Pelletization increases the density of planttissue. This increases the efficiency of the extraction process,decreases the extractors' volume and decreases the amount of solventsneeded for extraction. In addition, plant tissue pellets are relativelyeasy to handle. For example, there are little or no problems associatedwith dust or filtration.

[0126] As used herein, “drying” refers to the process of removing asubstantial amount (e.g., up to about 50%, up to about 75%, or up toabout 90%) of liquid or moisture in the plant tissue. In an alternativeembodiment, the drying process can remove up to about 95%, up to about99%, or up to about 100% of liquid or moisture in the plant tissue. Theliquid or moisture can typically include water. As such, the drying willeffectively remove at least a portion of water present in the planttissue. Prior to or subsequent to fragmentation, plant tissue can bedried. Such drying may increase the efficiency of the fragmentation,which can increase the efficiency of the extraction. The plant tissuecan be air-dried or dried at an elevated temperature with or withoutreduced pressure (i.e., in vacuo). Typically, the drying temperatureemployed is any suitable elevated temperature that will not lead todegradation or decomposition of the plant tissue or the componentstherein. Specifically, plant tissue can be dried in vacuo at an elevatedtemperature. Machines capable of drying plant tissue are known in theart and include an oven, or similar device, such as a rotating air drumdrier. The plant tissue can be dried at any suitable temperature. Forexample, the plant tissue can be dried above about 25° C., above about50° C., or above about 100° C. Additionally, the plant tissue can bedried for any suitable period of time. For example, the plant tissue canbe dried for more than about 10 minutes, for more than about 1 hour, orfor more than about 24 hours. Additionally, any suitable pressure can beemployed that does not lead to decomposition or degradation of the planttissue. The drying pressure typically can be from about 0.1 atmosphereto 2.5 atmospheres. Specifically, the drying pressure can be from about0.1 atmosphere to 1.0 atmosphere. More specifically, the drying pressurecan be from about 0.1 atmosphere to 0.75 atmosphere.

[0127] After the fragmented plant tissue is optionally dried andoptionally pelletized, it typically can be placed in an extractionvessel to be extracted. Any suitable extraction vessel can be employed.The extraction vessel will preferably be equipped with inlets andoutlets that can be opened and closed. The vessel will optionally becapable of being heated and/or pressurized. For smaller scaleextractions, the vessel can be, e.g., a soxhlet apparatus. Forcommercial scale (e.g., kilogram) extractions, the extraction vessel canbe a stainless steel tube or similar chamber, optionally attached to asolvent reservoir. Additionally, commercial scale (e.g., kilogram)extractions, the extraction vessel can be a vessel capable ofsupercritical fluid extraction (SFE).

[0128] The plant tissue can be packed to fill the capacity (or somefraction thereof) of the extraction vessel. The extent to which theextraction vessel can be filled with the packed, fragmented plant tissuewill vary depending upon the dimensions of the extraction vessel, theplant tissue, and the density to which the plant tissue can be packed.The fragmented plant tissue typically can be packed to any suitabledensity depending upon the extraction vessel and the scale of theextraction. Preferably, the plant tissue can be packed to a density ofabout 0.1 g/cm³ to about 1 g/cm₃. More preferably, the fragmented planttissue can be packed to a density of about 0.25 g/cm₃ to about 0.85g/cm₃ in the extraction vessel. More preferably, the fragmented planttissue can be packed to a density of about 0.5 g/cm³ to about 0.75g/cm³. However, the optimal extent to which the extraction vessel can bepacked with plant tissue and the density to which the plant tissue canbe packed can be easily determined by practitioners in the art.Additionally, the extraction vessel can be filled up to about 99% of thevolume, up to 90% of the volume, or up to about 80% of the volume.

[0129] Supercritical Fluid Extraction (SFE)

[0130] The second extraction solvent, the third extraction solvent, orcombination thereof, can employ supercritical fluid extraction.

[0131] Supercritical fluid extraction is an extraction wherein a fluidat a temperature and pressure above its critical point is employed; or afluid above its critical temperature, regardless of pressure, isemployed. Below the critical point, the fluid can coexist in both gasand liquid phases, but above the critical point there is only one phase.Equipment and techniques for carrying out supercritical fluid extractionare known to those skilled in the art. See, McHugh, M. And Krukonis, V.,Supercritical Fluid Extraction, 2nd ed, Butterworth-Heinemann, Boston,1994; Johnston, K. P., Penninger, J. M. L., Supercritical Fluid Scienceand Technology, ACS Symposium Series 406, American Chemical Society,Washington, D.C.; and Taylor, L.T., Supercritical Fluid Extraction, JohnWiley & Sons, New York, 1996.

[0132] In a supercritical fluid extraction, thermodynamic and transportproperties of supercritical fluid are a function of density, whichdepends strongly on the fluid's pressure and temperature. The densitymay be adjusted from a gas-like value of 0.1 g/ml to a liquid-like valueas high as 1.2 g/ml. Furthermore, as conditions approach the criticalpoint, the effect of temperature and pressure on density becomes muchmore significant. For example, increasing the density of supercriticalcarbon dioxide from 0.2 to 0.5 g/ml requires raising the pressure from85 atm to 140 atm (8.6 megapascals to 14.2 megapascals) at 158° F. (70°C.), but at 95° F. (35° C.) the required change is only from 65 atm to80 atm (6.61 Mpa to 8.1 Mpa).

[0133] As used herein, “fractional supercritical fluid extraction”(hereinafter “FSCFE”) is a multi-step procedure wherein thesupercritical fluid extraction is carried out at one temperature andpressure for a given period of time and is then carried out at one ormore other temperatures or pressures.

[0134] The efficiency of supercritical fluid extraction on a materialsuch as outer birch bark depends in part upon the size of the outerbirch bark pieces. Thus, the smaller the outer birch bark pieces, themore efficient the supercritical fluid extraction typically will be. Assuch, after fragmentation and prior to extraction, outer birch barkshreds may be further reduced in size with a Hammermill or suitablemeans. For example, a 15 horsepower 3B Junior Hammermill made by Jay BeeManufacturing, Inc can be used as illustrated in the Examples hereinbelow. The hammermill reduces large pieces of birch bark by beating thebark with pivoted hammers until the material is small enough to fallthrough a mesh.

[0135] For use in the processes of the present invention, the size ofouter birch bark shreds obtained after the Hammermill reduction istypically less than about 5 mm in diameter. Specifically, the shreds canbe less than about 3 mm in diameter. More specifically, the shreds canbe less than about 1 mm in diameter.

[0136] For use in the processes of the present invention, supercriticalfluid extraction can conveniently be carried out at a pressure of about1,000 psi to about 12,000 psi. It is appreciated that those skilled inthe art understand that higher pressures may enable faster extraction.In this case, it may be necessary to subsequently separate and purifythe product.

[0137] For use in the processes of the present invention, supercriticalfluid extraction can conveniently be carried out at a pressure of about750 psi to about 12,000 psi. Specifically, the pressure may be about1,000 psi to about 10,000 psi. More specifically, the pressure may beabout 4,000 psi to about 9,000 psi.

[0138] For use in the processes of the present invention, thetemperature of supercritical fluid extraction can conveniently be about0° C. to about 150° C. Specifically, the temperature can be about 25° C.to about 110° C. More specifically, the temperature can be about 45° C.to about 100° C.

[0139] In one specific embodiment, supercritical fluid extraction isperformed at a temperature of about 40° C. to about 90° C. and apressure of about 3,000 psi to about 10,000 psi.

[0140] Supercritical fluid extraction employs a solvent which possessesphysical properties suitable as a supercritical fluid. Suitable solventsinclude carbon dioxide, Xe, Freon-23, ethane, N₂O, SF₆, propane,ammonia, n-C₄H₁₀, (C₂H₅)₂O and the like.

[0141] The physical and environmentally friendly properties of carbondioxide make it particularly attractive as a solvent for supercriticalfluid extraction. Carbon dioxide is a major component of the atmosphereand is therefore relatively safe and abundant. In addition, carbondioxide is relatively inexpensive. Compared to most other suitablesolvents, carbon dioxide is environmentally friendly as it will not harmthe atmosphere at the quantities used in the methods of the invention.Moreover, carbon dioxide is non-flammable and non-explosive. Further,carbon dioxide leaves no substantial residue or remnant uponevaporation.

[0142] Carbon dioxide also possesses physical properties which enable itto change polarity over the temperature range and pressure rangenormally employed in supercritical fluid extraction. As a result, carbondioxide may act as a nonpolar solvent at one temperature and pressurebut may act as a polar solvent at another temperature and pressure. Byvarying the temperature and pressure, the solvent properties may bemodified. This allows for the isolation of more than one compound usinga single solvent system.

[0143] The solvent employed in supercritical fluid extraction may be asingle compound or may be a mixture of compounds. In addition, thesolvent may include an additive.

[0144] The non-acidic extract (i.e., the non-acidic compounds) willinclude relatively little or no acidic compounds therein. Typically, thenon-acidic extract will include less than about 25 wt. % acidiccompounds, less than about 10 wt. % acidic compounds, less than about 5wt. % acidic compounds, or less than about 1 wt. % acidic compounds.

[0145] The collected non-acidic extract can be purified by methods knownin the art such as, e.g., crystallization, chromatography, distillation,or a combination thereof. See in “Experiments in Organic Chemistry: FromMicroscale to Macroscale,” Jonathan S. Nimitz (New York: Prentice Hall,1990).

[0146] An optional adsorption process can be employed in the method ofthe present invention. Without an adsorption process, birch bark can beextracted employing the extraction procedures disclosed herein toprovide a betulin fraction may have a tan appearance. The color isprobably caused by aligomeric tannin admixtures, which are not acidicenough to be bound by Al(i-OPr)₃ in the extraction vessel. To adsorb thecolor causing compounds, adsorbents such as silica, aluminum oxide,calcium carbonate, calcium oxide, molecular sieves, ionic exchangeresins, amberlite, sephadex, sephacryl, polymeric adsorbents (diaion,ambersorb), cellulose, hydroapatite, and/or activated charcoal may beemployed. Preferably, the adsorbent for decoloration can be Al₂O₃.

[0147] After the non-acidic compounds are removed from the plant tissue,the acidic compounds, which underwent reaction with the basic componentto form salts, can optionally be removed. Discharging the second solventfrom the vessel and introducing a mixture of an acid in a third solventcan accomplish this.

[0148] The acidic extract (i.e., the acidic compounds) will includerelatively little or no non-acidic compounds therein. Typically, theacidic extract will include less than about 25 wt. % non-acidiccompounds, less than about 10 wt. % non-acidic compounds, less thanabout 5 wt. % non-acidic compounds, or less than about 1 wt. %non-acidic compounds.

[0149] The resulting acidic extract can be purified by methods known inthe art such as, e.g., crystallization, chromatography, distillation, ora combination thereof See “Experiments in Organic Chemistry: FromMicroscale to Macroscale,” Jonathan S. Nimitz (New York: Prentice Hall,1990).

[0150] The invention is further demonstrated in the following example.The example is for purposes of illustration and is not intended to limitthe scope of the present invention.

EXAMPLE I Selective Low-High Pressure Extraction of Pretreated OuterBirch Bark Pellets in a 3-Liter-Extraction Vessel

[0151] Introduction

[0152] A method for selectively isolating compounds from plant tissue,such as birch bark, includes the pretreatment of the plant tissue, e.g.,birch bark, selective extraction of the pretreated tissue, and theisolation and purification of non-acidic and acidic compounds, such asbirch bark triterpenes, which include betulin (a), lupeol (b), betulinicacid (c) and betulin-3-caffeate (d).

[0153] Birch bark extraction was performed using an apparatus (FIG. 1)purchased from Newport Scientific, Inc. (model: 46-19360-50 Hz). Theapparatus can be used for low and high-pressure operations (up to10,000.00 psi or 680 atm). The low-pressure pump (3) (KNF Flodos, Co.,CH-6210 Sursee) and distillation/recycling assembly (4, 5, 6, 8, 9 and10) are additional units for the apparatus. The apparatus may be usedwith organic solvents, such as i-propanol, ethanol, methylene chloride,toluene, and o-, p- and m-xylene, at temperatures from about 50° C. to200° C., as well as CO₂ supercritical extraction conditions. For CO₂supercritical extraction, it is necessary to use the diaphragm typecompressor, which may compress supercritical fluids, gas or liquid, to10,000 psi.

[0154] To extract and selectively isolate non-acidic and/or acidiccompounds from birch bark, the bark was first, shredded, ground,screened and pelletized (cylinder shaped pellets, b×h=1 mm×1 mm) asdescribed in WO 01 108885. The average density of the pellets was about0.5 to 0.6 g/ml. The pellets were dried in a drying hood at about 100°C. for about 10 hours before loading into an extraction vessel.

[0155] Step 1: Birch Bark Pellet Pretreatment Process

[0156] Approximately 1.2 kg of dry birch bark pellets were placed/loadedin a 20 L Rotor Evaporator (Buchi Rotavapor R-153). Approximately 60 g(1.5%) of Al(O-i-Pr)₃ in 4 L of p-xylene was added to a rotating vesselof the rotor evaporator at room temperature. The birch bark pellets andAl(O-i-Pr)₃ in p-xylene were rotated in the rotator vessel under normalpressure at 70° C. for about 2 hours. After 2 hours, the p-xylene wasevaporated at p=30-40 mm at a temperature of about 60-70° C. Followingevaporation, 1 L of p-xylene was added into the rotor evaporator. Thisadditional solvent was evaporated at p=30-40 mm at a temperature ofabout 60-70° C. The additional solvent and evaporation allowed for therelease of birch bark pellets from undesirable admixtures of i-propanol(see Equation 1 below). As a result of the pretreatment process, themajority of acidic compounds present in the birch bark were bound innon-soluble aluminum salts as demonstrated in Equation 1. Alcohols, suchas betulin and lupeol, are not acidic enough to be bound withAl(O-i-Pr)₃. The birch bark pellets maintained their cylinder/pelletform (b×h=1 mm×1 mm) throughout the pretreatment process.$\begin{matrix}{\left. {{XH} + {{Al}\text{(}i\text{-}{OPr}\text{)}_{3}}}\rightarrow{X - {{OAl}\text{(}i\text{-}{OPr}\text{)}_{2}} + {i\text{-}{PrOH}}} \right.\text{}{\text{XH}\text{-the~~acidic~~component~~of~~natural~~material}}} & {{Equation}\quad 1}\end{matrix}$

[0157] Step 2: The Process of Selective Extraction

[0158] After pretreatment, the pellets, about 1260 g, were loaded intoan extraction vessel for the selection extraction process. Theapparatus, including the parts listed in Table I, were assembled asshown in FIG. 1. The extraction system consisted of three major blocks:(1) the extraction vessel assembly; (2) the adsorption vessel assembly;and (4) the solvent regeneration assembly. The extraction vesselassembly (1) and adsorption vessel assembly (2) may maintain thepressure up to 10,000.00 psi (680 atm.). Such pressure levels may beused for CO₂ supercritical extraction. The extraction vessel (EV) (1)was equipped with a thermocouple (19.1), thermocouple temperaturecontroller (20.1), regulating valves (13.3, 13.6) and heaters (14). TheEV (1) was connected to the adsorption vessel (2) by stainless steeltubing, which was heated by heating tape (11). The adsorption vessel (2)was also equipped with a thermocouple (19.2), thermocouple temperaturecontroller (20.2), regulating valves (13.4, 13.7) and adsorption vesselheaters (15). The adsorption vessel (2) was connected to the distillingflask (4) through a valve (13). The distilling flask (4) was equippedwith a Wurtz adapter (8), thermometer (18) and stopper (7). Thehorizontal large condenser (6) was attached to adapter (8) and adapter(9). Adapter (9) was connected to the receiving flask (5). The pump (3)was attached through a coupling joint and PE tubing with a receivingflask (5). The pump assembly was equipped with a back loop system (12).A nitrogen tank was attached to a regulating valve (13.2). TABLE 2 Thelist of equipment for pretreated birch bark pellets extraction No. Nameof Equipment Quantity  1 Extraction vessel, 3L, stainless steel 1  2Adsorption Vessel, 1L, stainless steel 1  3 Low pressure pump 1  4Distilling flask, vert, 3N, 45/50, 5L 1  5 Receiving flask, vert, 3N,45/50, 3L 1  6 Condenser large horizontal, 45/50, 20″ 1  7 Stopper,45/50 2  8 Wurtz distillation adapter, 45/50 1  9 Distillation adapter,45/50 1 10 Condenser small vertical 1 11 Heating tape 2 12 Back loopsystem 1 13 Regulating valve 9 14 EV heater 4 15 AV heater 2 16 DFheater 1 17 RF heater 1 18 Thermometer 1 19 Thermocouples 2 20-24 Flatbottom 4L-flasks 5

[0159] A. Packing of the Extraction 3 L-vessel (1)

[0160] The stainless steel bottom dispersion plate (with 1 mm diameterholes) was placed at the bottom of extraction vessel (1). Filter paper,cotton filter and one or more additional filter papers were placed onthe dispersion plate. Approximately 1260 g of pretreated birch barkpellets were placed in the extraction vessel through the wide-neckfunnel. Filter paper and the dispersion plate were then placed on top ofthe pellets. The extraction vessel (1) was filled with 1500 ml ofp-xylene. The extraction vessel was closed/sealed with a gasket-equippedlid. The thermocouple (19.1) was inserted and the lid of vessel (1) wasclosed with eight bolts. The thermocouple screw was also tightened. Theregulating valves (13.3) and (13.6) were attached to the top and thebottom of the extraction vessel. The stainless steel tubing wasconnected with the out-coming from the pump, to the top-regulating valve(13.1). The stainless steel tubing, wrapped with heating tape, was alsoconnected to the bottom-regulating valve (13.9).

[0161] B. 2.2. Packing the Adsorption Vessel (2)

[0162] Filter paper, cotton filter and another filter paper were placedon the dispersion plate at the bottom of the adsorption vessel (2).Approximately 300 g of aluminum oxide (active acidic, activity #1, 7—230Mesh ASTM) with 0.6 L of p-xylene was poured carefully and stirred intothe adsorption vessel (4). Filter paper was placed on top of thealuminum oxide, then 400 g of dry calcinated sand, followed by anotherfilter paper and finally a dispersion plate. The adsorption vessel (4)was closed with a lid, the thermocouple (19.2) was inserted and then thelid was tightened with twelve bolts. After tightening the lid, thethermocouple (19.2) was tightened with a screw. The regulating valve(13.4) was attached to the lid of the adsorption vessel. Stainless steeltubing was connected, out-coming from the extraction vessel, to thetop-regulating valve (13.4). Stainless steel tubing was also connectedfrom the bottom of the adsorption vessel (2) to the regulating valve(13.7).

[0163] C. Assembly of the Solvent Distillation System (4)

[0164] The coupling joint was connected to the stainless steel tubing,out-coming from the regulating valve (13.7). A 5-liter distilling flask(4) was placed into the distilling flask heater (16) and the couplingjoint inserted. The Wurtz distillation adapter (8) was placed into theneck of the distilling flask. A thermometer (18) was installed into theWurtz adapter (8). A stopper (7) was placed into the central neck of thedistilling flask. The distillation adapter (9) was attached to thereceiving flask and attached to a large condenser (6). The top of thelarge condenser (6) was adjusted to the Wurtz distillation adapter (8).The drying tube was placed on top of the small condenser (10). Theextraction vessel out-coming tubing was connected to the receiving flaskthrough the coupling joint.

[0165] D. Extraction Process

[0166] About 3500 ml of p-xylene was added to the distilling flask. Thedistilling flask heater was turned on. The solvent was warmed up to itsboiling point. The temperature on the heating tape (11) and the heaters(14, 15) was set to about 130° C. The extraction vessel (1), adsorptionvessel (2) and tubing had an average temperature of about 120-130° C.Valves 13.2 and 13.8 were closed, while valves 13.1, 13.3, 13.4, 13.6,13.7 and 13.9 were open. When the receiving flask (5) was half filledwith freshly distilled p-xylene, the pump was turned on. The pumpingspeed was equal to the speed of distillation (about 65-70 ml/min). Theextraction was continued for about 2.5 hours and then the pump wasturned off. All valves were then closed except for valve (13.5). About1500 ml of p-xylene was distilled. The distilling flask heater wasturned off. The extract (1^(st)-fraction) was transferred from vessel(4) to the 4 L-flat bottom flask (20). The 1^(st)-fraction, incrystallization flask (20), was placed in a freezer to enable betulincrystallization at a temperature of about −5 C. The total volume of the1^(st)-fraction extract was approximately 2300 ml.

[0167] Approximately 1800 ml of p-xylene and 50 ml of acetic acid wasadded to the distilling flask (5). About 30 ml of acetic acid was addedto the receiving flask (5). The distilling flask heater was turned on.The solvent was heated to its boiling point. The heating tape (15) andheaters (14, 15) were turned on. The temperature of the vessels andtubing was maintained at approximately 100° C. Valves 13.2 and 13.8 wereclosed, while valves 13.1,13.3, 13.4,13.6, 13.7 and 13.9 were open. Pump(3) was turned on. The pumping speed was equal to the speed ofdistillation (about 65-70 ml/min). The extraction was continued for 2.5hours and the pump (3) was turned off. All valves, except valve 13.5,were closed. Approximately 1500 ml of solvent was distilled into thereceiving flask (5). The distilling flask heater was turned off. Thecontents from the distilling flask (4) (2^(nd)-fraction) weretransferred into the 3 L-flat bottom flask (21). Valves 13.1 and 13.8were closed, while vales 13.2, 13.3, 13.4, 13.6, 13.7, and 13.9 wereopen. The nitrogen flow valve (13.2) was opened (200 ml of N₂/min) for30 minutes and the residue extract was collected in the distillingflask. The flown extract was transferred to flask (21). The total volumeof the 2^(nd)-fraction extract was about 2400 ml. The distilled solventwas transferred from the receiving vessel (5) to the 3 L flat bottomflask for solvent recycling (22).

[0168] The temperature in the extraction system was maintained around100C. 5 L of 95% i-propanol-5% water solvent was prepared and 1500 mlwas loaded in the receiving flask (5) and 3500 ml into the distillingflask (4). The distilling flask heater was then turned on. The solventwas heated to its boiling point. Valves 17.2 and 17.8 were closed, whilevalves 13.1, 13.3, 13.4, 13.6, 13.7 and 13.9 were opened. The pump wasthen turned on. The pumping speed was maintained at a level equal to thespeed of distillation (about 60-70 ml/min). The extraction continued for2.5 hours, after which, the pump was turned off and all valves, exceptthe valve 13.5, were closed. Approximately 1500 ml of solvent wasdistilled into the receiving flask. The distilling flask heater wasturned off. The extract (3^(rd)-fraction) was transferred from thedistilling flask (4) into the flat bottom flask (23). Valves 13.1 and13.8 were closed, while valves 13.2, 13.3, 13.4, 13.6, 13.7, and 13.9were open. The nitrogen flow valve (13.2) was opened (200 ml/min) for 30min. The flown extract was collected in vessel (4) and transferred toflask (23). The total volume of the 3^(rd)-fraction extract wasapproximately 2200 ml. The distilled solvent from the receiving vessel(5) was transferred into the flask for solvent recycling (24).

[0169] Step 3: Birch Bark Triterpenes Isolation

[0170] A. Betulin Isolation

[0171] The 1^(st) fraction solution (2.3 L from flask 20) was cooleddown at a temperature of about −5° C. in a freezer for about 4 hours.The white precipitate was then filtered using a Buchner funnel 600 mland Bunzen flask 4 L. The crystals in the funnel were washed withp-xylene (2×100 ml) at a temperature of approximately 5-10° C. The whitecrystals were dried in vacuum at a temperature of about 90° C. up to theconstant weight. About 145 g of 98%⁺ pure betulin was obtained (gaschromatography (GC), high performance liquid chromatography (HPLC)). Theyield of betulin from starting birch bark pellets was about 12%.

[0172] B. Lupeol Isolation

[0173] The liquid portion (about 2500 ml) remaining after betulinfiltration was evaporated under reduced pressure in a rotor evaporator.About 32-35 g of dry material was crystallized twice with dry acetone.The white crystals of lupeol were then dried in vacuum up to theconstant weight. Approximately 14.6 g of 95%⁺ pure lupeol was obtained(GC, HPLC). The yield of 95%⁺ pure lupeol from starting birch barkpellets was about 1.2%.

[0174] C. Betulinic Acid Isolation

[0175] The 2^(nd)-fraction solution (2.4 L from flask 22) was evaporatedunder reduced pressure to obtain 27 g of a dark colored betulinic acidfraction containing about 60% betulinic acid, as determined by HPLCanalysis. The fraction containing approximately 60% of betulinic acid,approximately 27 g, was boiled with 400 ml of isopropanol and 4.2 g ofsodium hydroxide for about 2 hours. The solution was cooled to atemperature of about 40° C. and the isopropanol was evaporated underreduced pressure. The remaining solid material was transferred into theSoxlet apparatus and extracted with 300 ml of p-xylene for 3 hours. Thesolid material was dried under vacuum and transferred to a 1 L beaker towhich 700 ml of water was added and stirred at about 1000 rpm for about1 h. The precipitate was filtered and washed with about 100 ml of 5%sodium hydroxide (pH >10). The remaining solid material was transferredinto a 1 L beaker to which 300 ml of water was added and acidified withapproximately 5% hydrochloric acid (pH=5.2-5.5). The precipitate wasfiltered and dried on a filter. The dried precipitate was thencrystallized twice from i-propanol. Approximately 12 g of 95%⁺ purebetulinic acid was obtained (GC, HPLC). The yield of betulinic acid fromstarting birch bark pellets was approximately 1.0%.

[0176] C. Betulin 3-Caffeate Isolation

[0177] The 3^(rd)-fraction solution (2.2 L, flask 23) of thecrystallization flask was evaporated under reduced pressure.Approximately 47 g of a brown betulin-3-caffeate fraction, whichcontains 48% betulin-3-caffeate by HPLC analysis, was obtained. About 15g of betulin-3-caffeate was obtained (93%⁺ pure, HPLC, nuclear magneticresonance (NMR)) by column chromatography on silica withether/hexane=6/4. The yield of betulin-3-caffeate from starting birchbark pellets was about 1.25%.

[0178] All publications, patents and patent applications areincorporated herein by reference. While in the foregoing specificationthis invention has been described in relation to certain preferredembodiments thereof, and many details have been set forth for purposesof illustration, it will be apparent to those skilled in the art thatthe invention is susceptible to additional embodiments and that certainof the details described herein may be varied considerably withoutdeparting from the basic principles of the invention.

What is claimed is:
 1. A method for selectively extracting one or morenon-acidic compounds from plant tissue in the presence of one or moreacidic compounds, the method comprising: (a) contacting a mixture of abasic component and a first solvent with the plant tissue to immobilizethe acidic compound as a salt on the plant tissue; and (b) contactingthe plant tissue with a second solvent suitable to remove the one ormore non-acidic compounds; thereby effectively providing a solutioncomprising the one or more non-acidic compounds.
 2. The method of claim1, wherein the basic component comprises an alcoholate.
 3. The method ofclaim 2, wherein the alcoholate is an aluminum alcoholate.
 4. The methodof claim 3, wherein the aluminum alcoholate is aluminum iso-propoxide.5. The method of claim 3 wherein the aluminum alcoholate is aluminumethoxide or aluminum methoxide.
 6. The method of claim 1 wherein thebasic component comprises an amine of formula NR₃, wherein each R isindependently hydrogen, (C₁-C₁₂)alkyl, aryl (e.g., phenyl), or arylalkyl(e.g., benzyl), wherein each alkyl, aryl (e.g., phenyl), or arylalkyl(e.g., benzyl) can be optionally substituted on carbon with one or morehydroxy, halo, or —N(R_(b))₂; wherein R_(b) is H, (C₁-C₆)alkyl, aryl(e.g., phenyl), or arylalkyl (e.g., benzyl).
 7. The method of claim 1wherein the basic component comprises a heterocycle.
 8. The method ofclaim 7 wherein the heterocycle is pyridine, morpholine, piperidine,pyrrole, or pyrrolidine; each optionally subtituted on any suitablecarbon with oxo, hydroxy, sulfo, (C₁-C₄)alkyl, (C₁-C₄)hydroxyalkyl, or—N(R_(b))₂, wherein R_(b) is H or (C₁-C₄)alkyl, or on nitrogen with(C₁-C₄)alkyl or (C₁-C₄)hydroxyalkyl.
 9. The method of claim 1 whereinthe basic component comprises an alkaline earth metal hydroxide.
 10. Themethod of claim 9 wherein the basic component is NaOH, KOH, LiOH,Mg(OH)₂, Ca(OH)₂, or a mixture thereof.
 11. The method of claim 1wherein the basic component comprises an alkaline earth metal oxide. 12.The method of claim 1 wherein the basic component comprises an alkalineearth metal carbonate or an alkaline earth metal bicarbonate.
 13. Themethod of claim 12 wherein the basic component is Na₂CO₃, K₂CO₃, KNaCO₃,Li₂CO₃, CaCO₃, MgCO₃, or a mixture thereof.
 14. The method of claim 1wherein the basic component comprises an alkaline earth metal sulfite oralkaline earth metal sulfide.
 15. The method of claim 1, wherein thefirst solvent and the second solvent are each independently anoptionally substituted aromatic compound, an optionally substitutedheterocyclic compound, an optionally substituted cyclic compound, anoptionally substituted linear or branched compound, or combinationthereof, wherein suitable substituents include (C₁-C₆)alkyl, hydroxyl,halo, cyano, nitro, oxo, thioxo, amino, carboxyl, or combinationsthereof.
 16. The method of claim 1, wherein the first solvent and thesecond solvent are each independently isopropanol, ethanol, methanol,methylene chloride, toluene, o-xylene, m-xylene, p-xylene, carbondioxide, Xe, Freon-23, ethane, N₂O, SF₆, propane, ammonia, n-C₄H₁₀,(C₂H₅)₂O, or a combination thereof.
 17. The method of claim 1, whereinthe first solvent, the second solvent, or a combination thereof,comprises an additive.
 18. The method of claim 17 wherein the additiveis methanol; ethanol; 1-propanol; 2-propanol; 1-hexanol; 2-methoxyethanol; tetrahydrofuran; 1,4-dioxane; acetonitrile; dichloromethane;ammonia; chloroform; propylene carbonate; N,N-dimethylacetamide;dimethyl sulfoxide; formic acid; water; carbon disulfide; acetone;propane; toluene; hexanes; pentanes; o-xylene; m-xylene; p-xylene;toluene; or a combination thereof.
 19. The method of claim 1 wherein theplant tissue comprises bark, roots, leaves, flowers, needles, bulbs,berries, rhizomes, rootstocks, stems, seeds, or any combination thereof.20. The method of claim 1 wherein the plant tissue comprises Taxusyunnanesis bark, yew tree needles, Echinacea spp. root, Ginkgo bilobaroot bark, Ginkgo biloba leaves; Allium sativum bulbs, Valerianaofficinalis root, Panax ginseng root, Aloe vera leaves, Vacciniummacrocarpon berries, Eleutherococcus senticosus root, Eleutherococcussenticosus rhizome, Eleutherococcus senticosus stems, Eleutherococcussenticosus leaves, Piper methysticum rootstock, dill seeds, kola nutseeds, cinchona red bark, chinchona yellow bark, or a combinationthereof.
 21. The method of claim 1 wherein the one or more non-acidiccompounds comprises lupeol, betulin, taxol, echinacea extract, valerianroot extract, ginkgolide A, ginkgolide B, ginkgolide C, bilobalide,garlic extract, ginseng extract, aloe gel, barbaloin, cranberry extract,eleutheroside A, eleutheroside B, eleutheroside C, eleutheroside D,eleutheroside E, eleutheroside G, kava extract, dill seed oil, kolaextract, quinoline alkoloids, or a combination thereof.
 22. The methodof claim 1 wherein the one or more non-acidic compounds obtained in theselective extraction comprises less than about 5 wt. % acidic compounds.23. The method of claim 1, further comprising contacting the planttissue with an acid in a third solvent, to neutralize the salt and toremove the one or more acidic compounds from the plant tissue.
 24. Themethod of claim 23 wherein the acid comprises hydrochloric acid,hydrobromic acid, sulfuric acid, phosphoric acid, acetic acid, formicacid, or a combination thereof.
 25. The method of claim 23, wherein thethird solvent comprises an optionally substituted aromatic compound, anoptionally substituted heterocyclic compound, an optionally substitutedcyclic compound, an optionally substituted linear or branched compound,or combination thereof, wherein suitable substituents include(C₁-C₆)alkyl, hydroxyl, halo, cyano, nitro, oxo, thioxo, amino,carboxyl, or combinations thereof.
 26. The method of claim 23, whereinthe third solvent comprises isopropanol, ethanol, methanol, methylenechloride, toluene, o-xylene, m-xylene, p-xylene, carbon dioxide, Xe,Freon-23, ethane, N₂O, SF₆, propane, ammonia, n-C₄H₁₀, (C₂H₅)₂O, or acombination thereof.
 27. The method of claim 23, wherein the thirdsolvent comprises an additive.
 28. The method of claim 27 wherein theadditive is methanol; ethanol; 1-propanol; 2-propanol; 1-hexanol;2-methoxy ethanol; tetrahydrofuran; 1,4-dioxane; acetonitrile;dichloromethane; ammonia; chloroform; propylene carbonate;N,N-dimethylacetamide; dimethyl sulfoxide; formic acid; water; carbondisulfide; acetone; propane; toluene; hexanes; pentanes; o-xylene;m-xylene; p-xylene; toluene; or a combination thereof.
 29. The method ofclaim 1, wherein the one or more acidic compounds comprises betulinacid, betulin-3-caffeate, tannin, phenol, caffeic acid, cichoric acid,valerenic acid, isovaleric acid, flavonoid, quercetin, kaempferol,catechin, lignin, shikimic acid, succinic acid, amino acid, nicotinicacid, pantothenic acid, anthraquinone, acidic galactan, benzoic acid,quinic acid, malic acid, citric acid, hippuric acid, phenolic acid,ferulic acid, chlorogenic acid, norsolorinic acid, fatty acids, tartaricacid, dicaffeate, cinnamic acid, or a combination thereof.
 30. Themethod of claim 29 wherein the one or more acidic compounds obtainedfrom the selective extraction comprises less than about 5 wt. %non-acidic compounds.
 31. A method for selectively extracting one ormore non-acidic compounds from plant tissue in the presence of one ormore acidic compounds, the method comprising: (a) contacting the planttissue with an aluminum alkoxide in a first solvent to immobilize theacidic compound as a salt on the plant tissue; and (b) contacting theplant tissue with a second solvent suitable to remove the one or morenon-acidic compounds; thereby effectively providing a solutioncomprising the one or more non-acidic compounds; wherein the one or morenon-acidic compounds comprises lupeol, betulin, taxol, paclitaxel,echinacea extract, valerian root extract, ginkgolide A, ginkgolide B,ginkgolide C, bilobalide, garlic extract, ginseng extract, aloe gel,barbaloin, cranberry extract, eleutheroside A, eleutheroside B,eleutheroside C, eleutheroside D, eleutheroside E, eleutheroside G, kavaextract, dill seed oil, kola extract, quinoline alkoloids, or acombination thereof; and wherein the one or more acidic compoundscomprises betulin acid, betulin-3-caffeate, tannin, phenol, caffeicacid, cichoric acid, valerenic acid, isovaleric acid, flavonoid,quercetin, kaempferol, catechin, lignin, shikimic acid, succinic acid,an amino acid, nicotinic acid, pantothenic acid, anthraquinone, acidicgalactan, benzoic acid, quinic acid, malic acid, citric acid, hippuricacid, phenolic acid, ferulic acid, chlorogenic acid, norsolorinic acid,a fatty acid, tartaric acid, dicaffeate, cinnamic acid, or a combinationthereof.
 32. The method of claim 31, further comprising treating theplant tissue with an acid in a third solvent to provide a solutioncomprising the one or more acidic compounds.
 33. A method forselectively extracting lupeol, betulin, or a combination thereof frombirch bark in the presence of betulinic acid, betulin-3-caffeate, or acombination thereof, the method comprising: (a) contacting the birchbark with an aluminum alkoxide in a first solvent comprising xylenethereby effectively immobilizing the betulinic acid, betulin-3-caffeate,or combination thereof as a salt on the birch bark; and (b) contactingthe birch bark with a second solvent suitable to remove the lupeol,betulin, or combination thereof.
 34. The method of claim 33, furthercomprising treating the birch bark with an acid in a third solvent toprovide the betulinic acid, betulin-3-caffeate, or combination thereof.35. A composition of matter comprising the one or more non-acidiccompounds as prepared in any one of claims 1-34.
 36. A composition ofmatter comprising the one or more acidic compounds as prepared in anyone of claim 23, 32, or
 34. 37. Taxol obtained by the process ofcontacting a mixture of a basic component and a first solvent with Taxusyunnanesis bark to immobilize one or more acidic compounds on the Taxusyunnanesis bark and contacting the Taxus yunnanesis bark with a secondsolvent suitable to remove the taxol, to effectively provide a solutioncomprising taxol comprising less than about 5 wt. % of tannins, fattyacids, phenols, or a combination thereof.
 38. Betulin obtained by theprocess of contacting a mixture of a basic component and a first solventwith birch bark to immobilize one or more acidic compounds on the birchbark and contacting the birch bark with a second solvent suitable toremove the betulin, to provide a solution comprising betulin comprisingless than about 5 wt. % of betulinic acid, betulin-3-caffeate, or acombination thereof.